Stage-specific control of oligodendrocyte survival and morphogenesis by TDP-43

Generation of oligodendrocytes in the adult brain enables both adaptive changes in neural circuits and regeneration of myelin sheaths destroyed by injury, disease, and normal aging. This transformation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes requires processing of distinct mRNAs at different stages of cell maturation. Although mislocalization and aggregation of the RNA-binding protein, TDP-43, occur in both neurons and glia in neurodegenerative diseases, the consequences of TDP-43 loss within different stages of the oligodendrocyte lineage are not well understood. By performing stage-specific genetic inactivation of Tardbp in vivo, we show that oligodendrocyte lineage cells are differentially sensitive to loss of TDP-43. While OPCs depend on TDP-43 for survival, with conditional deletion resulting in cascading cell loss followed by rapid regeneration to restore their density, oligodendrocytes become less sensitive to TDP-43 depletion as they mature. Deletion of TDP-43 early in the maturation process led to eventual oligodendrocyte degeneration, seizures, and premature lethality, while oligodendrocytes that experienced late deletion survived and mice exhibited a normal lifespan. At both stages, TDP-43-deficient oligodendrocytes formed fewer and thinner myelin sheaths and extended new processes that inappropriately wrapped neuronal somata and blood vessels. Transcriptional analysis revealed that in the absence of TDP-43, key proteins involved in oligodendrocyte maturation and myelination were misspliced, leading to aberrant incorporation of cryptic exons. Inducible deletion of TDP-43 from oligodendrocytes in the adult central nervous system (CNS) induced the same progressive morphological changes and mice acquired profound hindlimb weakness, suggesting that loss of TDP-43 function in oligodendrocytes may contribute to neuronal dysfunction in neurodegenerative disease.


Introduction
The distribution of myelin in the adult brain is not static, as new oligodendrocytes continue to be generated in response to changes in experience, such as enhanced sensory input and intense motor learning (Hughes et al., 2018;Xiao et al., 2016). As a result, oligodendrocyte lineage cells exist in a developmental continuum throughout life, consisting of oligodendrocyte precursor cells (OPCs) that maintain their density through proliferation (Hughes et al., 2013), and cells in the process of maturing from premyelinating to myelinating oligodendrocytes. This ongoing oligodendrogenesis is necessary to prolong the period of circuit maturation (Benamer et al., 2020), enable adaptive changes in myelination (Gibson et al., 2014), and restore myelin lost through aging or demyelinating injuries (Orthmann-Murphy et al., 2020;Bacmeister et al., 2020). The transition from progenitor to myelinforming oligodendrocyte requires profound transcriptional changes, in which myelin-associated gene networks are activated and mRNAs are transported to distal processes to direct sites of axon ensheathment and membrane compaction (Müller et al., 2013). These transcriptional and posttranscriptional processes are orchestrated by RNA-binding proteins. However, the factors that coordinate key developmental transitions within the oligodendrocyte lineage to enable OPC homeostasis and myelin dynamics are not well understood.
TDP-43 (TAR DNA-binding protein 43 kDa) is a DNA/RNA-binding protein that serves as a master regulator of RNA metabolism, controlling transcriptional integrity, post-transcriptional stability, and localization of mRNAs (Warraich et al., 2010). Within the nucleus, TDP-43 prevents incorporation of cryptic exons into mature mRNA by binding to UG-rich motifs within intronic sequences (Ling et al., 2015). Loss of TDP-43 in neurons results in missplicing of pre-mRNAs, resulting in gain and loss of function alterations in the landscape of expressed proteins that influence their structure, function, and longevity (Ling et al., 2015;Donde et al., 2019;Ma et al., 2021;Brown et al., 2021). In many neurodegenerative diseases, including frontotemporal dementia, Alzheimer's disease (AD), amyotropic lateral sclerosis (ALS), and multiple sclerosis (MS), cytoplasmic mislocalization and aberrant aggregation of TDP-43 have been observed in affected regions of the CNS (Neumann et al., 2006;Ling et al., 2015;Sun et al., 2017;Masaki et al., 2020;Salapa et al., 2020). Moreover, mutations in TARDBP are responsible for both familial and sporadic forms of ALS (Kapeli et al., 2017;Ince et al., 2011), suggesting that altered mRNA processing by TDP-43 may contribute to cellular pathology and functional impairment in both sporadic and inherited forms of neurodegenerative disease. Although neurons have been the primary focus of research into TDP-43-associated pathologies, nuclear exclusion, and cytoplasmic aggregation of TDP-43 also occur in glial cells in neurodegenerative diseases, such as MS (Neumann et al., 2006;Masaki et al., 2020;Rohan et al., 2014), suggesting that glial cell dysfunction could impair both OPC homeostasis and remyelination. However, much less is known about the role of TDP-43 in mRNA metabolism or the impact of its loss within oligodendroglia at distinct stages of maturation.
Genetic deletion of TDP-43 in mice results in embryonic lethality due to impaired expansion of inner cell mass during early embryogenesis (Sephton et al., 2010), suggesting that it is essential for cell survival. However, selective deletion experiments indicate that the effects of TDP-43 are cell type specific and diverse (Jeong et al., 2017), reflecting differences in transcriptional state. In neurons, conditional inactivation of TDP-43 induces impairment in neuronal function and progressive neurodegeneration (Donde et al., 2019;Iguchi et al., 2013;Yang et al., 2014;Wu et al., 2019). Conditional knockout of Tardbp in microglia enhanced expression of NF-κB and NLRP3, leading to a more activated phenotype characterized by enhanced phagocytosis (Zhao et al., 2015;Paolicelli et al., 2017), while loss of TDP-43 in astrocytes promoted their transition to a reactive A1 phenotype associated with neuronal injury (Peng et al., 2020;Walker et al., 2014). In Schwann cells, genetic deletion of Tardbp did not affect their survival or myelinic potential, but impaired formation and maintenance of paranodal junctions (Chang et al., 2021), while deletion from both newly generated oligodendrocytes and Schwann cells induced necroptosis of oligodendrocytes and early mortality (Wang et al., 2018). These results indicate that TDP-43 regulates transcriptional integrity of different genes based on cellular context, highlighting the need to define TDP-43 functions within different cellular states. As oligodendroglia consist of both proliferating progenitors and cells in distinct states of maturation, their unique transcriptional profiles may render them differentially sensitive to loss of TDP-43.
Here, we used in vivo targeted genetic manipulations of the oligodendrocyte lineage to define the consequences of TDP-43 loss of function in the mouse brain. OPCs were the most vulnerable to this manipulation, as selective deletion of TDP-43 from OPCs resulted in their rapid death, progressing from the corpus callosum to the overlying cortical gray matter. However, this progenitor depletion was transient, as homeostatic proliferation of OPCs that retained TDP-43 expression led to rapid repopulation and restoration of their normal density. Unexpectedly, the consequences of deletion of TDP-43 in oligodendrocytes were dependent on their stage of maturation. Early deletion resulted in the progressive degeneration of mature oligodendrocytes, leading to seizures and premature lethality. In contrast, late deletion largely spared oligodendrocytes, and mice survived without seizures, indicating that TDP-43 may be dispensable within mature oligodendrocytes. Strikingly, both early and late manipulation of TDP-43 in oligodendrocytes led to profound morphological changes, accompanied by thinner and fewer myelin sheaths, as well as progressive, inappropriate wrapping of neuronal somata and blood vessels. Conditional removal of TDP-43 from oligodendrocytes in the adult CNS induced similar morphological changes and was associated with profound hindlimb weakness. Differential transcriptional analysis of oligodendrocytes revealed that loss of TDP-43 led to missplicing of RNAs encoding key genes involved in oligodendrocyte morphogenesis and myelination, changes that may contribute to abnormal neuronal activity and neurodegeneration in diverse neurological diseases.

Selective deletion of TDP-43 from OPCs induces their progressive loss
Oligodendrocytes are derived from an abundant, widely distributed population of lineage restricted progenitors, termed OPCs, that maintain their density through local homeostatic proliferation (Hughes et al., 2013). The dynamic nature of these cells may render them particularly susceptible to environmental and genetic perturbations. To determine how loss of TDP-43 affects OPCs in vivo, we selectively deleted Tardbp from OPCs in young adult Pdgfra-CreER;Tardbp fl/fl ;RCE (PDGFRα-TDP43 cKO) mice, by administering tamoxifen at P90 and then collecting brain tissue 2 weeks later (P90 + 14) ( Figure 1A). Inclusion of the RCE transgene (Rosa-CAG-LSL-EGFP) allows the visualization of the morphology of Cre-recombined cells (Sousa et al., 2009). Immunolabeling for EGFP and NG2, a proteoglycan highly expressed by OPCs, revealed that this manipulation induced recombination in most OPCs (EGFP+NG2+/NG2+ cells: 94.75 ± 0.75%, n = 4) ( Figure 1B), resulting in near complete depletion of OPCs from the corpus callosum, while those in the overlying cortical gray matter were preserved ( Figure 1C,E; Figure 1D, panel i) (OPCs/mm 3 : Corpus callosum 14 dpi, Control = 6910 ± 201; cKO = 987 ± 201, n = 3, p < 0.001, one-way ANOVA with Tukey's multiple comparisons test). However, when the interval between tamoxifen administration and sacrifice was increased to 30 days (P90 + 30), OPCs in gray matter were also depleted ( Figure 1C,E; Figure 1D, panel ii) (OPCs/mm 3 : Cortex 30 dpi, Control = 5247 ± 236; cKO = 811 ± 187, n = 3, p < 0.001, one-way ANOVA with Tukey's multiple comparisons test). Remarkably, OPCs in white matter had repopulated by this time, and highly polarized OPCs were visible migrating out of the corpus callosum into the gray matter ( Figure 1C, red arrowhead; Figure 1D, panel iii; Figure 1E). When the interval between tamoxifen administration and sacrifice was increased by another 30 days (P90 + 60), OPCs had regained their normal density and tiled distribution throughout gray and white matter ( Figure 1C,E). As expected, this repopulation was associated with enhanced proliferation of OPCs, visible by increased Ki-67 immunoreactivity in PDGFRα-TDP43 cKO ( Figure 1F; Figure 1-figure supplement 1A). Despite this extensive death of OPCs, there were no signs of astrogliosis, as assessed by glial fibrillary acidic protein (GFAP) immunoreactivity ( Figure 1-figure supplement 1B), in accordance with the tolerance to normal turnover of these cells (Hughes et al., 2013). This lack of glial reactivity in response to widespread loss of OPCs was also described in a previous OPC ablation study using diphtheria toxin (Birey et al., 2015), suggesting that OPC death does not induce reactive changes in astrocytes or microglia. Deletion of Tardbp in P180 mice also resulted in a robust loss of OPCs in the corpus callosum within 14 days (P180 + 14) (Figure 1-figure supplement 1C), indicating that the dependence on TDP-43 extends into the adult CNS. Together, these results show that TDP-43 is required for the survival and homeostasis of oligodendrocyte progenitors. The online version of this article includes the following figure supplement(s) for figure 1:
Deletion of TDP-43 from premyelinating oligodendrocytes impairs oligodendrocyte survival OPCs undergo a profound series of structural and physiological changes as they differentiate into myelinating oligodendrocytes, a transition enabled by activation of distinct gene regulatory networks expressed at different stages of their maturation (Xu et al., 2020;He et al., 2017). To determine if postmitotic oligodendrocytes are similarly dependent on TDP-43, we deleted Tardbp from these cells using two constitutive Cre lines which differ in their timing of expression during oligodendrocyte maturation: Mog iCre , which has been used extensively to manipulate mature oligodendrocytes (Buch et al., 2005;Larson et al., 2018; Figure  . Single-cell RNA-Seq results indicate that Mobp expression is robust at the premyelinating oligodendrocyte stage, whereas expression of Mog does not increase substantially until the myelinforming stage (Marques et al., 2016;Hrvatin et al., 2018;Figure 2-figure supplement 3). Gene expression analysis of oligodendroglia at these stages revealed that many mRNAs are differentially expressed, including Gadd45a, which participates in DNA methylation, and Opalin (Tmem10) that has been shown to promote oligodendrocyte differentiation (Barreto et al., 2007;de Faria et al., 2019;Figure 2-figure supplement 4), suggesting that the consequences of TDP-43 may be distinct. Abolishing TDP-43 expression at these different stages resulted in very different outcomes. Mice in which TDP-43 was constitutively removed from premyelinating oligodendrocytes (Mobp-iCre;Tardbp fl/ fl ;RCE) (Mobp-TDP43 KO) began to exhibit spontaneous seizures at ~P110 ( Figure 2B, red arrowhead; Video 1) and 100% died prematurely by P180 ( Figure 2B, dark orange line), similar to the phenotype exhibited by Cnp Cre ;Tardbp fl/fl mice (Wang et al., 2018), in which recombination occurs in OPCs, early postmitotic oligodendrocytes, and Schwann cells in the PNS (Lappe-Siefke et al., 2003;Chang et al., 2021;Tognatta et al., 2017). In contrast, mice in which TDP-43 was deleted from mature oligodendrocytes (Mog iCre ;Tardbp fl/fl ;RCE) (Mog-TDP43 KO) did not exhibit spontaneous seizures or other gross behavioral abnormalities and survived as well as controls ( Figure 2B, blue lines).
In contrast to the extensive demyelination and oligodendrocyte necroptosis observed in Cnp Cre ;Tardbp fl/fl mice (Wang et al., 2018), there was no change in the density of ASPA+ mature oligodendrocytes in the motor cortex of Mobp-TDP43 KO or Mog-TDP43 KO mice at P30, P90, or P180 (end stage) ( Figure 2C,D). However, degenerating oligodendrocytes can be rapidly replaced through differentiation of OPCs , obscuring extensive cell loss. As remaining OPCs proliferate to maintain their density, higher rates of OPC proliferation are associated with oligodendrocyte degeneration . In accordance with the different behavioral phenotypes of the Mobp-TDP43 KO and Mog-TDP43 KO mice, OPCs also exhibited strikingly different behaviors in these animals. Early deletion of TDP-43 in premyelinating oligodendrocytes (Mobp-TDP43 KO) was associated with a dramatic increase in Ki-67+ OPCs at P90 ( Figure 2E,F) (Ki-67+ OPCs/mm 3 : Mobp-TDP43 Control = 71 ± 45; Mobp-TDP43 KO = 941 ± 121, n = 4, p < 0.001, one-way ANOVA with Tukey's multiple comparisons test), whereas deletion of TDP-43 from mature oligodendrocytes (Mog-TDP43 KO) was not associated with a significant change in OPC proliferation ( Figure 2E,F) (Ki-67+ OPCs: Mog-TDP43 Control = 24 ± 24; Mog-TDP43 KO = 212 ± 89, n = 4, p > 0.05, one-way ANOVA with Tukey's multiple comparisons test). Moreover, there was an increase in the number of newly formed oligodendrocytes in Mobp-TDP43 KO, as assessed by the emergence of cells that expressed lncOL1 ( Figure 2-figure supplement 4A), a long noncoding RNA that is transiently expressed in premyelinating oligodendrocytes (He et al., 2017), suggesting that there is an increased turnover of oligodendrocytes. Astrogliosis (visible through the increase in GFAP immunoreactivity) was also prominent in Mobp-TDP43 KO mice (Figure 2-figure supplement 4B, C), further supporting the conclusion that early deletion of TDP-43 leads to degeneration and subsequent regeneration of oligodendrocytes. Together, these results indicate that there is a critical period for TDP-43 function during oligodendrocyte maturation, with deletion at an early stage leading to progressive oligodendrocyte degeneration, seizures, and premature death, consequences that are avoided when oligodendrocytes mature beyond this stage. In addition to thinner myelin sheaths, the number of myelinated axons was also reduced in both oligodendrocyte TDP-43 knockouts ( Figure 3E,F). Morphological reconstructions of individual oligodendrocytes and their associated myelin sheaths revealed that Mog-TDP43 KO oligodendrocytes tended to exhibit less myelin basic protein (MBP) immunoreactivity per process (revealed by EGFP labeling), indicative of decreased myelin production ( Figure 3-figure supplement 3). These results indicate that despite achieving a mature myelinating stage, the total myelin output of TDP-43-deficient oligodendrocytes is ultimately lower (summarized in Figure 3-figure supplement 4).
In peripheral nerves, loss of TDP-43 from Schwann cells results in complete loss of paranodal junctions and a 50% decrease in conduction velocity (Chang et al., 2021). Immunocytochemical analysis revealed that TDP-43-null oligodendrocytes also frequently exhibited abnormal nodes of Ranvier (NOR). While normal NOR exhibit focal localization of βIV spectrin at the node flanked by Caspr within the paranodes (

Oligodendrocytes without TDP-43 undergo profound morphological changes
To define the progressive structural changes exhibited by TDP-43-deficient oligodendrocytes, we examined their morphologies in Mobp-TDP43 KO and Mog-TDP43 KO mice using EGFP (expressed from the conditional RCE transgene) (Sousa et al., 2009). Histological analysis of statistical difference between the samples at any given timepoint (one-way ANOVA with Tukey's multiple comparisons test, n = 3, n.s. p value >0.05).
The online version of this article includes the following figure supplement(s) for figure 2:      motor cortex in these mice revealed that oligodendrocyte density gradually increased from P30 to P180 in both control and mutant (KO) mice ( Figure 2D); however, while EGFP was largely restricted to oligodendrocyte somata in controls, due to the limited cytoplasm in myelin sheaths, mice with TDP-43-deficient oligodendrocytes exhibited a dramatic increase in EGFP fluorescence in the surrounding parenchyma at P90 and P180 (Figure 4-figure supplement 1). This morphological change in oligodendrocytes was observed throughout the brain, as well as the optic nerve and spinal cord (Figure 4figure supplement 2), suggesting that this phenotype is fully penetrant in the CNS. Repetitive in vivo two photon imaging of individual oligodendrocytes in the motor cortex of Mobp-TDP43-RCE KO mice over 2 weeks revealed that these aberrant structures are highly dynamic (Figure 4-figure supplement 3; Video 2), indicating that these normally highly stable cells exhibit enhanced growth when TDP-43 is absent. High-resolution imaging revealed that much of this additional EGFP fluorescence arose from thick cytoplasmic processes of oligodendrocytes that often ended in circular or cufflike structures ( Figure 4A,B). Immunostaining for NeuN revealed that circular structures arose from inappropriate wrapping of neuronal cell bodies ( Figure 4C,D; Video 3). These abnormal associations were not rare events and were visible in single coronal brain sections immunostained for NeuN and EGFP ( Figure 4-figure supplement 4).
As the cuff-like structures are reminiscent of astrocyte endfeet (Gundersen et al., 2014), we labeled blood vessels with GS-Isolectin, revealing that EGFP+ oligodendrocyte processes also colocalized with these tubular structures ( Figure 4E). To determine if these structures were capillaries, we used in vivo two photon imaging in living Mobp-TDP43-RCE KO mice to visualize both oligodendrocyte processes and capillaries after a retroorbital injection of dextran-rhodamine. With 4D imaging, it was possible to follow the cytoplasmic processes from individual oligodendrocytes, revealing that they extended unbranched processes to ensheath rhodamine-labeled capillaries ( Figure 4F). Histological analysis revealed that regions of capillaries surrounded by oligodendrocyte cuffs exhibited reduced aquaporin four immunoreactivity ( Figure 4-figure supplement 5A), a protein restricted to astrocyte endfeet (Gundersen et al., 2014), suggesting that these oligodendrocyte wraps may displace or alter the physiology of astrocytes at capillaries. Remarkably, we also found that some TDP-43 KO oligodendrocytes exhibited immunoreactivity to Ki-67 ( Figure 4-figure supplement 5B The online version of this article includes the following figure supplement(s) for figure 3:           oligodendrocytes and myelin throughout the CNS.

Conditional deletion of TDP-43 from oligodendrocytes in the adult CNS induces abnormal morphological changes and motor discoordination
The results above indicate that constitutive deletion of TDP-43 from myelinating oligodendrocytes induces a profound morphological transformation, and when removed early in their maturation, results in oligodendrocyte degeneration, seizures, and death. Although oligodendrocytes continue to be generated in the adult CNS, most oligodendrocytes are produced in the first two postnatal months (Bergles and Richardson, 2015), indicating that these changes primarily manifest in developing tissue. To determine if loss of TDP-43 function in oligodendrocytes in the mature brain evokes comparable changes, we generated Mobp-iCreER mice, which allow selective Cre-dependent recombination in oligodendrocytes in the adult CNS (see Materials and methods; Figure 2-figure supplement 2). When tamoxifen was administered to P90 Mobp-iCreER;Tardbp fl/fl ;RCE mice and analyzed 30 days later (P90 + 30), ASPA+ oligodendrocytes were present in the motor cortex with morphologies comparable to controls (Mobp-iCreER;Tardbp +/+, fl/+ ) ( Figure 5A,B). However, when TDP-43 mutant mice were analyzed 90 days later (P90 + 90), the majority of oligodendrocytes exhibited hypertrophy, as well as cytoplasmic ensheathment of neuronal cell bodies and capillaries ( Figure 5B, yellow arrowhead), indicating that TDP-43 is also required to maintain oligodendrocyte structure in the adult CNS. However, the behavioral consequences of TDP-43 deletion in the mature CNS were distinct, as these mice exhibited progressive motor problems, including limp tails, hindlimb clasping ( Figure 5C, Video 4), and hindlimb weakness ( Figure 5D), but not seizures or premature lethality. We utilized the clinical scoring method established for experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, and found that Mobp-TDP43 cKO developed significant motor weakness within 90 days after Cre recombination ( Figure 5E). Although this transformation of oligodendrocytes did not lead to degeneration of motor neurons ( Figure 5-figure supplement 1), the spinal cord exhibited enhanced GFAP immunoreactivity ( Figure 5F,G), indicative of widespread inflammation, which has been shown to impair motor function without loss of spinal motor neurons (Wang et al., 2018). Thus, adult-onset loss of TDP-43 function in oligodendrocytes, which may occur in disease and injury states (Wiesner et al., 2018;Sun et al., 2017), triggers phenotypes commonly observed in mouse models of ALS, Parkinson's disease (PD), and MS (Wong and Martin, 2010;Lieu et al., 2013;Gharagozloo et al., 2021).

TDP-43 regulates splicing and represses cryptic exon incorporation in oligodendrocytes
TDP-43 is a highly conserved, multifunctional protein that regulates key aspects of RNA processing. Previous studies have shown that loss of nuclear TDP-43 function, through genetic deletion or diseaseinduced mislocalization, is sufficient to cause aberrant incorporation of intronic sequences (cryptic exons) into mature mRNA that can alter protein expression and function (Ling et al., 2015;Sun et al., 2017;Donde et al., 2019). Due to the unique complement of RNAs present in different cell types and differences in intron-exon boundaries between organisms, these changes are both cell type and species specific (Jeong et al., 2017). To define the molecular consequences of TDP-43 loss of function in oligodendrocytes, we used fluorescence-activated cell sorting (FACS) to selectively isolate oligodendrocytes from the cortex of P30 Mobp-TDP43-RCE KO and Mog-TDP43-RCE KO mice (and controls) prior to the onset of phenotypic changes ( Figure 4A; Figure 3-figure supplement 1), and performed bulk mRNA sequencing (RNA-Seq) to determine gene expression changes ( Figure 6A). In both mouse lines, hundreds of genes were differentially expressed, comprising 3-6% of the mouse genome ( Figure 6B; Figure 6-figure supplement 1), consistent with the magnitude of changes  observed in other cell types and the high incidence of UG repeats recognized by TDP-43 in the genome (Humphrey et al., 2017). The differential gene expression profiles and gene ontology analysis were also distinct between the mutant mouse lines ( Figure 6B; Figure 6-figure supplement 2), with early deletion resulting in changes in mRNA processing and cell localization, and latter deletion resulting in changes in regulation of gene expression and metabolic processes. Analysis of the top 1% of the highest expressed genes enriched in oligodendrocytes, many of which are involved in oligodendrocyte development and myelination, revealed that cryptic exons were often incorporated in their mRNAs ( Figure 6F). These included Ermn (Ermin), an oligodendrocyte-specific cytoskeletal protein that regulates process branching and sheath extension (Brockschnieder et al., 2006). TDP-43 loss resulted in the emergence of a novel cryptic exon in intron 2 of the Ermn transcript ( Figure 6C). RT-PCR validation using whole mouse brain indicated that this aberrant splicing generated novel splice isoforms, arising from cryptic exon incorporation, exon extension, and truncation ( Figure 6D) that may lead to both gain and loss of function. Total Ermin protein was significantly lower in Mobp-TDP43-RCE KO brains compared to controls ( Figure 6E), and immunohistochemical staining revealed that Ermin was no longer detected in oligodendrocyte somata in these animals ( Figure 6figure supplement 3). Thus, TDP-43 deficiency leads to an early missplicing of Ermn mRNA, resulting in a partial loss of functional protein. These results indicate that progressive loss of TDP-43 from oligodendrocytes in the adult CNS leads to altered splicing of key regulators of oligodendrocyte growth and morphogenesis, disrupting myelin patterns, inducing gliosis and impairing neuronal function.

Discussion
New oligodendrocytes are generated in the adult CNS to modify circuits and restore myelin lost through injury or disease (Gibson et al., 2014;Orthmann-Murphy et al., 2020). This oligodendrogenesis requires transformation of highly dynamic, proliferative progenitors into stable, postmitotic oligodendrocytes that exhibit extraordinary longevity (Tripathi et al., 2017). The profound structural and functional changes experienced by oligodendrocyte lineage cells are orchestrated by a family of RNA-binding proteins that ensure transcripts are spliced correctly and delivered to their appropriate location within the cell prior to translation (Müller et al., 2013). Cellular stress or direct mutation of these genes can lead to mislocalization and progressive loss of their function, contributing to cellular pathology in diverse neurodegenerative diseases (Kapeli et al., 2017;Purice and Taylor, 2018), but the consequences of these effects within oligodendroglia are not well understood. As oligodendrocyte lineage cells continue to exist in a dynamic developmental continuum in the adult CNS, these changes may influence progenitor homeostasis, lineage progression, cellular survival, and function, depending on the stage at which gene function is disrupted.
To genetically interrogate the role of TDP-43 within different stages of oligodendrocyte maturation, we employed four different Cre and CreER mouse lines: (1) Pdgfra-CreER, (2) Mobp-iCre, (3) Mog iCre , and (4) Mobp-iCreER, allowing Tardbp inactivation at discrete stages within the oligodendrocyte lineage in both the developing and mature CNS. Bulk RNA-Seq of purified oligodendrocytes from Mobp-iCre and Mog iCre mouse lines showed that this approach was able to target different stages of oligodendrocyte development. In particular, oligodendrocytes labeled by Mobp-iCre showed higher expression of genes enriched in newly formed oligodendrocytes, such as Gpr37 and Mag, whereas those labeled by Mog iCre had higher expression of genes enriched in myelin-forming and mature oligodendrocytes,

Ncam1
Alternative splicing Neural cell adhesion molecule    (Figure 6-figure supplement 4). Therefore, these two mouse lines, in combination with Pdgfra-CreER mice to manipulate OPCs (Kang et al., 2010), allowed specific interrogation of TDP-43 function at distinct developmental stages, revealing a differential requirement for TDP-43 in cell survival and structural maintenance as OPCs transform into myelinating oligodendrocytes ( Figure 6-figure supplement 5).

Homeostasis promotes rapid recovery after OPC depletion
Inducible genetic deletion of Tardbp within OPCs resulted in their rapid degeneration, with loss first detected in the white matter of the corpus callosum 2 weeks after tamoxifen exposure, followed closely by OPC depletion in cortical gray matter, demonstrating that these progenitors require TDP-43 for survival. OPCs represent the largest population of actively dividing cells in the brain outside the subventricular and subgranular germinal zones (Dawson et al., 2003), and maintain a constant density through homeostatic proliferation, in which neighboring cells are induced to divide to replace cells lost through death or differentiation (Hughes et al., 2013). Proliferative cells in many tissues are particularly vulnerable to perturbations in RNA processing, as they undergo metabolic reprogramming requiring extensive transcriptional reorganization (Petasny et al., 2021). Thus, death of only a few proliferating OPCs upon removal of TDP-43 is likely to induce a cascading cellular depletion, in which neighboring cells (also TDP-43 deficient) are induced to proliferate but then die, inducing further proliferation of remaining cells and continuing until the entire population of TDP-43-deficient OPCs is depleted. The higher basal rate of OPC proliferation in white matter, reflecting their higher rate of differentiation (Baracskay et al., 2002;Viganò et al., 2013), may be responsible for more rapid loss of OPCs in this region.
Despite decimation of OPCs after TDP-43 deletion, they were restored to their original density in both gray and white matter within 30 days. Because Tardbp was not inactivated in all OPCs (the proportion limited by the efficiency of tamoxifen-induced recombination), surviving cells that retain TDP-43 are expected to proliferate to reconstitute the population. Consistent with this hypothesis, all OPCs after 30 days of recovery were EGFP negative (Figure 1-figure supplement 1D), indicating that they did not experience Cre activity. These results imply that focal loss of OPCs arising from deficits in TDP-43 should not lead to persistent depletion. It is also possible that the regenerated OPCs were derived from the neural progenitor pool in the subventricular zone, as previously described in the context of remyelination (Xing et al., 2014). Indeed, OPCs have been shown to be exceptionally resistant to perturbations in their distribution and density (Xing et al., 2021;Dang et al., 2019), and rapidly restore their numbers after mass differentiation following demyelination in mice (Baxi et al., 2017;Penderis et al., 2003). Nevertheless, reductions in OPC density have been observed in some human MS lesions (Chang et al., 2000), suggesting that other environmental changes, such as persistent inflammation or gliosis, which may induce TDP-43 mislocalization, may impede their homeostasis and impair their ability to detect demyelinated axons and generate new oligodendrocytes.

Stage-dependent oligodendrocyte survival in the absence of TDP-43
The differentiation of OPCs into mature, myelinating oligodendrocytes is associated with dramatic structural and functional changes, in which highly motile OPCs stabilize, elaborate processes, and select targets for myelination (Kirby et al., 2006;Almeida et al., 2011). Previous studies of Tardbp Source data 1. Raw, cropped, and annotated images of the western blot of mouse-Ermin (red; IRDye 680RD) and rabbit-GAPDH (green; IRDye 800CW).       (Wang et al., 2018;Chang et al., 2021), which recombines in the late progenitor and early premyelinating stages, indicate that loss of TDP-43 during this transition period leads to RIPK1-mediated necroptosis of oligodendrocytes (Wang et al., 2018). We used two constitutive Cre lines (Mobp-iCre and Mog iCre ) expressed at different stages (early and late) of the maturation of postmitotic oligodendrocytes to determine whether the effects of TDP-43 loss depend on their state of differentiation. In contrast to TDP-43-deficient OPCs, oligodendrocytes were generated, and myelin sheaths were formed in both lines despite inactivation of Tardbp. As the half-life of TDP-43 in vitro was measured to be 12-34 hr Lee et al., 2011), it is possible that sufficient residual TDP-43 RNA/protein remains from the OPC stage to enable these initial transitions. Although oligodendrocytes and myelin were generated, TDP-43-deficient oligodendrocytes formed sheaths with fewer wraps and overall fewer axons were myelinated, similar to that observed after Cnp Cremediated deletion (Wang et al., 2018). Reconstructions of individual oligodendrocytes at this stage revealed that fewer sheaths were formed per cell on average, indicating that the overall myelin output of these newly formed oligodendrocytes is compromised in the absence of TDP-43. However, the ultimate outcome of these manipulations was distinct, with early deletion (using Mobp-iCre) resulting in oligodendrocyte degeneration, seizures, and premature death by 6 months of age. This phenotype is similar to, but not as severe as that observed in Cnp Cre ;Tardbp fl/fl mice, in which mature oligodendrocytes, OPCs, premyelinating oligodendrocytes, and Schwann cells were genetically manipulated (Wang et al., 2018;Chang et al., 2021), indicating that disruption of oligodendrocyte TDP-43 levels is sufficient to enhance neuronal excitability and induce premature death.
Although oligodendrocyte density remained stable after Mobp-iCre-mediated Tardbp inactivation, reactive gliosis occurred throughout the CNS and there was an increase in premyelinating oligodendrocytes (lncOL1+) and enhanced proliferation of OPCs, indicative of oligodendrocyte degeneration, similar to the phenotypic changes observed after inducible deletion of oligodendrocytes in the adult CNS (Traka et al., 2015;Oluich et al., 2012). Previous studies have shown that subtler manipulations of mature oligodendrocytes, such as reduction in expression of the inwardly rectifying potassium channel K ir 4.1 (Larson et al., 2018) or deletion of the lactate transporter MCT1 (Philips et al., 2021), is sufficient to alter neuronal excitability, highlighting possible functional consequences of TDP-43 loss from these myelinating cells.
Unexpectedly, inactivation of Tardbp from oligodendrocytes at a later developmental stage (with Mog iCre ) led to a strikingly different outcome: oligodendrocytes survived, OPC proliferation was not enhanced and there was no gliosis. Correspondingly, these animals also did not exhibit seizures or premature death. Bulk RNA-Seq transcriptional analysis of oligodendrocytes with early and late deletion, even prior to the phenotypic changes in Mobp-iCre mice (P30), revealed that pathways involved in gene expression, cellular localization, cell adhesion, and metabolism were affected. Further, differential gene expression analysis of early, premyelinating and myelinating oligodendrocytes from obtained through single-cell RNA sequencing (Hrvatin et al., 2018) revealed that different transcription factors, such as Mycl, and transporters, such as MCT1 (Slc16a1), change in gene expression as oligodendrocytes mature (Figure 2-figure supplement 4). Thus, rapid changes in transcriptional activity during cellular maturation can profoundly alter the impact of TDP-43 depletion, with deficits occurring earlier in maturation leading to more severe outcomes.
The phenotypes observed in response to deletion of TDP-43 from newly generated oligodendrocytes, including subsequent seizures and premature death, may also reflect developmental changes in neural circuits, as this manipulation inactivated Tardbp during a critical phase of brain maturation. Indeed, deletion of TDP-43 from oligodendrocytes in the adult CNS (using Mobp-iCreER) led to distinct pathological changes, such as hindlimb weakness, hindlimb clasping, and limp tail, phenotypes not observed with developmental Tardbp inactivation, and these mice did not exhibit seizures or premature death. Although these distinct phenotypes may arise from differences in the proportion of oligodendrocytes affected, they mirror functional differences observed in neurodevelopmental and neurodegenerative diseases. In neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia, abnormal myelination and altered density of oligodendrocytes have been correlated with neural hyperexcitability and circuit dysfunction (Phan et al., 2020;Fields, 2008). In contrast, oligodendrocyte turnover and demyelination in the adult CNS have been shown to be accompanied by inflammation and neuronal death in neurodegenerative diseases, such as ALS and AD Desai et al., 2010;Behrendt et al., 2013). These results suggest that the consequences of TDP-43 loss within the oligodendrocyte lineage depends not only on their stage of maturation, but also the environment in which it occurs, with progressive loss in the adult CNS sufficient to impair motor coordination, a hallmark of neurodegenerative diseases such as ALS.

Aberrant target ensheathment in the absence of TDP-43
In vitro studies indicate that oligodendrocytes have an intrinsic ability to wrap structures of appropriate caliber (>0.4 µm), including inert nanofibers and fixed axons (Rosenberg et al., 2008;Mei et al., 2013;Lee et al., 2012). However, oligodendrocytes in vivo exhibit exquisite target specificity, forming myelin exclusively around axons of appropriate diameter, while avoiding similarly sized dendrites and other nonneuronal structures, such as glial cell processes and capillaries. Moreover, after an initial period of remodeling, the array of myelin sheaths formed by each oligodendrocyte is extremely stable, in terms of position and length along axons. Deletion of TDP-43 from oligodendrocytes either early or late in their maturation resulted in striking, progressive changes in oligodendrocyte morphology, including extension of new processes and wrapping of inappropriate targets, such as neuronal somata and capillaries. Inappropriate myelination of neuronal somata has been observed after deletion of adhesive proteins (e.g., JAM2) from neurons (Redmond et al., 2016), expression of a mutant form of a cell adhesion protein (e.g., Cadm4) in oligodendrocytes (Elazar et al., 2019), or overexpression of MBP (Almeida et al., 2018). Similar aberrant myelination has been recently described in MS lesions (Neely et al., 2022), where TDP-43 mislocalization has also be observed (Masaki et al., 2020). The morphological changes exhibited by TDP-43-deficient oligodendrocytes suggests that they revert to a progrowth state (Grinspan et al., 1993;Lee et al., 2019), allowing them to overcome repulsive cues or lack of adhesion that normally prevents wrapping of nonaxonal structures.
Loss of TDP-43 leads to aberrant RNA splicing TDP-43 maintains the integrity of the cellular transcriptome by regulating splicing of pre-mRNAs and suppressing incorporation of cryptic exons. By efficiently binding to UG microsatellite repeats within intronic sequences, TDP-43 ensures that spliceosomes skip over these regions during normal splicing (Ling et al., 2015). As the specificity of TDP-43 targets primarily depend on the location of these UG tandem repeats, TDP-43-dependent cryptic exons are both cell type and species specific (Jeong et al., 2017;Ling et al., 2015). By performing RNA sequencing of TDP-43-deficient oligodendrocytes purified from the brain, we uncovered a novel set of cryptic exons in mRNAs that encode proteins involved in oligodendrogenesis and myelination, changes that resulted from novel cassette insertion, exon extension, and alternative splicing. In case of Ermin, a cytoskeletal protein involved in oligodendrocyte morphogenesis (Brockschnieder et al., 2006), cryptic exon incorporation led to a pronounced decrease in protein expression; however, the phenotypic changes induced by loss of TDP-43 are not the same as that observed in Ermn KO mice (Wang et al., 2020;Ziaei et al., 2020). Aberrant splicing of Ermin mRNA is predicted to lead to a truncated N-terminal peptide (~amino acid 1-110) that may produce a dominant negative effect. Moreover, loss of TDP-43 was not selective for Ermin, but led to aberrant splicing of numerous myelin and oligodendrocyte genes ( Figure 6F). Nfasc, which is misspliced in TDP-43-deficient Schwann cells (Chang et al., 2021), was not alternatively spliced in TDP-43-null oligodendrocytes, further confirming the cell-type-specific actions of TDP-43. The combination of gain and loss of function of the resultant proteins likely contribute to complex phenotype exhibited by these mutant oligodendrocytes.
Oligodendrocyte and myelin abnormalities have been reported in many neurodegenerative diseases known to present with TDP-43 proteinopathy Masaki et al., 2020;Zhang et al., 2019). Although cytoplasmic aggregates of TDP-43 are not always present, nuclear clearance of TDP-43 and resulting loss of function is sufficient to cause dramatic alterations in pre-mRNA splicing . Further investigation of RNA splicing changes in oligodendrocytes in different neurodegenerative diseases may reveal unique pathological signatures associated with disease states that may help predict progression and identify novel therapeutic approaches for reducing the impact of impaired TDP-43 activity.

Animal care and use
Both female and male mice were used for experiments and randomly assigned to experimental groups. Mice were maintained on a 12 hr light/dark cycle, housed in groups no larger than 5, and food and water were provided ad libitum. All animal experiments were performed in strict accordance with protocols approved by the Animal Care and Use Committee at Johns Hopkins University. All experiments were initially performed with a sample size of 3 (biological replicates) for quantification and statistical analyses, and power analysis was then performed to determine the minimum sample size to achieve sufficient statistical power to detect the effect of TDP-43 loss in different transgenic mouse lines at different timepoints. The following transgenic mouse lines were used in this study: Pdgfra-CreER (Kang et al., 2010) Tardbp floxed (Chiang et al., 2010) RCE (Rosa-CAG-LSL-EGFP) (JAX MMRRC stock #32038) (Sousa et al., 2009) Mobp-iCre Mobp-iCreER Mog iCre (Buch et al., 2005) Mobp-EGFP (Hughes et al., 2018) CreER Induction To induce Tardbp genetic deletion and EGFP expression in the Pdgfra-CreER;Tardbp floxed ;RCE and Mobp-iCreER;Tardbp floxed ;RCE mice, P90 or P180-240 mice were injected with tamoxifen (Sigma) dissolved in sunflower seed oil (Sigma) and administered by intraperitoneal injection daily for five consecutive days at a dose of 100 mg/kg body weight. Mice were perfused 14, 30, 60, and 90 days from the last day of tamoxifen injection.

Cranial windows and in vivo two-photon microscopy
Cranial windows were prepared as previously described (Orthmann-Murphy et al., 2020). Briefly, 4-to 8-week-old mice were anesthetized with isoflurane (induction, 5%; maintenance, 1.5-2%, mixed with 0.4-1 l/min O 2 ), and their body temperature was maintained at 37°C with a thermostat-controlled heating plate. The skin over the right hemisphere was removed and the skull cleaned. A 3 × 3 mm region of skull over motor cortex was removed using a high-speed dental drill. A piece of cover glass (VWR, No. 1) was placed in the craniotomy and sealed with VetBond (3 M), then dental cement (C&B Metabond) and a custom metal plate with a central hole was attached to the skull for head stabilization. In vivo imaging sessions began 3 weeks after cranial window procedure in order for the surgery-induced inflammation to subside. During imaging sessions, mice were anesthetized with isoflurane and immobilized by attaching the head plate to a custom stage. Images were collected using a Zeiss LSM 710 microscope equipped with a GaAsP detector using a mode-locked Ti:sapphire laser (Coherent Ultra) tuned to 920 nm.

Immunohistochemistry
Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially first with 15-20 ml room temperature PBS (pH 7.4) followed by 10 ml ice-cold 4% paraformaldehyde (PFA in 0.1 M phosphate buffer, pH 7.4). CNS tissues were dissected (e.g., optic nerve, brain, and spinal column) and postfixed in 4% PFA at 4°C for 4-6 or 12-20 hr depending on the antibodies to be used for immunohistochemistry. The tissues samples were transferred to 30% sucrose solution (in PBS, pH 7.4) for cryoprotection for at least two overnights before sectioning. Tissue samples were frozen in TissueTek and sectioned at 35 μm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Tissue sections were collected into a 24-well plate with PBS with 0.1% NaN 3 and kept in 4°C until use. Free-floating sections were preincubated in blocking solution (10% normal donkey serum, 0.3% Triton X-100 in PBS, pH 7.4) for 1 or 2 hr at room temperature, then incubated for 24-48 hr at 4°C or room temperature in primary antibody. Secondary antibody incubation was performed at room temperature for 2-4 hr. Sections were mounted on slides with Aqua Polymount (Polysciences). Images were acquired using a confocal laser-scanning microscope (Zeiss LSM 510 Meta; Zeiss LSM 800; Zeiss LSM 880). The following primary antibodies were used in this study:

Transmission electron microscopy (TEM) and G ratio quantification
Samples were perfuse fixed with room temperature PBS and 'Karlsson-Schultz' buffer (2.5% glutaraldehyde, 4% formaldehyde and 0.5% NaCl in phosphate buffer pH 7.3) and postfixed at 4°C for two overnights. Subsequent TEM processing was performed according to a previously described protocol (Weil et al., 2019). For G ratio analysis 10 random micrographs covering an area of ~180 µm 2 each were taken at a magnification of ×6000 using a LEO912 transmission electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany) with an on-axis 2k CCD camera (TRS, Moorenweis, Germany). For random selection of axons the images were overlaid with a grid and axons on grid crossings were analyzed with a minimum of 100 axons per animal. For G ratio determination the area of the axon and the corresponding fiber including the myelin sheath were measured using Fiji/ImageJ (Schindelin et al., 2012) and the diameters derived assuming circular areas. The G ratios were calculated as a ratio between axonal diameter and total fiber diameter.

Image processing and analysis
Image stacks and time series were analyzed using FIJI/ImageJ. For figure presentation, image brightness and contrast levels were adjusted for clarity. Density and G ratio quantifications were performed by a blinded observer. Longitudinal image stacks were registered using FIJI plugin 'Correct 3D Drift' (Parslow et al., 2014;Orthmann-Murphy et al., 2020). Quantification of abnormal wrapping was performed on maximum intensity z-projection images. Wrapping of neuronal soma was determined by EGFP+ processes wrapping around NeuN+ neuronal somata, whereas that of blood vessels was determined by the 'tube'-like structures made by EGFP+ oligodendrocyte processes.

Structural analysis of individual cortical oligodendrocytes
Cortices of Mog iCre ;Tardbp floxed ;Mobp-EGFP mice were flatmounted between glass slides, postfixed in 4% PFA for 12 hr at 4°C and transferred to 30% sucrose solution (in PBS, pH 7.4). Tissue was stored at 4°C for 48+ hr before sectioning. Flatmounted hemicortex were extracted, frozen in TissueTek, and sectioned at 40 µm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Free-floating immunohistochemistry against MBP and EGFP was performed on the first section of the flatmount, which consists of the layer I of the cerebral cortex, as described previously (Orthmann-Murphy et al., 2020). High-resolution images were acquired using a confocal laser-scanning microscope (Zeiss LSM 880). All processes originating from the cell body, branch points, and individual myelin sheaths stained with EGFP and MBP were traced in FIJI using Simple Neurite Tracer (Longair et al., 2011).
A smoothing function was used on the traced segments prior to length calculation to reduce tracing artifacts.

Clinical EAE behavioral scoring
Clinical EAE behavioral scores were obtained in a masked manner based on the preacquired videos of the animals, using the established standard scoring from 1 to 5; 0 = no signs of disease; 1 = loss of tail tonicity; 2 = loss of tail tonicity and mild paralysis of hindlimbs; 3 = paralysis of hindlimbs; 4 = hindlimbs paralysis and mild paralysis of forelimbs; and 5 = complete paralysis or death (Gharagozloo et al., 2021).

Statistical analysis
Statistical analyses were performed with GraphPad Prism 7 or Excel (Microsoft). All technical replicates (e.g., different brain sections for IHC) for each sample were averaged as a single data point. Significance was determined using one-way ANOVA test with Tukey's correction for multiple comparisons or unpaired, two-tailed Student's t-test. Each figure legend otherwise contains the statistical tests used to measure significance and the corresponding significance level (p value). n represents the number of animals (biological replicates) used in each experiment. Data are reported as mean ± standard error of the mean and p value <0.05 was considered statistically significant. RNA isolation, cDNA library preparation, and sequencing RNA was isolated from FACS purified oligodendrocytes (~5000 cells/sample) using QIAGEN RNeasy Micro Kit following the manufacturer's instructions. cDNA was synthesized by using QIAGEN Quanti-Tect Reverse Transcription Kit. Due to low amount of cDNA, Nugen Single-cell RNAseq kit was used to generate cDNA libraries for 100 bp paired-end sequencing on Illumina HiSeq (70-100 million paired reads/sample).

Analysis of bulk RNA-Seq data
Reads were aligned to the latest mouse mm10 reference genome using the STAR spliced read aligner with default parameters. Total counts of read fragments aligned to known gene regions within the mouse mm10 RefSeq reference annotation were used as the basis for quantification of gene expression. Fragment counts were derived using HTS-seq program using mm10 Ensembl transcripts as the model. Various QC analyses were conducted to assess the quality of the data and to identify potential outliers (e.g., the level of mismatch rate, mapping rate to the whole genome, repeats, chromosomes, and key transcriptomic regions). Principal component analysis correction was performed to correct for batch effects and differences in sample RNA integrity number. Differentially expressed genes were identified using EdgeR Bioconductor package and ranked based on adjusted p values (false discovery rate) of <0.05.

RT-PCR
RNA was isolated from whole mouse brain tissue using ReliaPrep RNA Tissue Miniprep System (Promega) following the manufacturer's instructions. cDNA was synthesized by using QIAGEN Quan-tiTect Reverse Transcription Kit. RT-PCR primers against normal Ermn and cryptic exon-specific Ermn transcripts were designed using Benchling and Primer3.

Western blotting
Whole mouse brain tissue was homogenized in 1.5 ml Eppendorf tube with a handheld homogenizer in Pierce RIPA buffer (Thermo Fisher) with Sodium Orthovanadate (Vanadate). After centrifugation at 4°C, supernatant was collected, and the protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher). 50 μg of protein was loaded into each lane of Mini-Protean TGX gels (BioRad) and ran at 120 V for 1.5 hr. Protein was transferred onto Immobilon-FL PVDF Membrane (Millipore) at 100 V for 1.5 hr. Intercept (TBS) Blocking Buffer (Li-Cor) was used for blocking, and the membrane was incubated in primary antibody solution at 4°C overnight with gentle shaking. Mouse-Ermin (Mab160; raised against Ermin residues 1-261) and Rabbit-GAPDH (Cell Signaling) primary antibodies were used. IRDye 680RD Goat anti-Mouse IgG (H + L) (Li-Cor) and IRDye 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor) were used as secondary antibodies for imaging on Odyssey Imaging Workstation (Li-Cor). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Additional files • Transparent reporting form